Dysfunctional cholesterol homeostasis is involved in the etiology of cardiovascular and other diseases. While much is known about extracellular cholesterol transport in plasma lipoproteins, the molecular mechanisms of intracellular cholesterol trafficking are not as well understood. A major remaining question in cellular cholesterol homeostasis concerns its transit through the endosomal vesicle recycling system. The lysosomal storage disease Niemann-Pick C (NPC) provides a window into this critical step. NPC disease is characterized by the accumulation of LDL-derived cholesterol in the late endosomal/lysosomal compartment (LE/LY). Caused by a deficiency in either of two LE/LY proteins, NPC1 or NPC2, the similarities in cellular and clinical phenotypes of NPC1 and NPC2 diseases suggest that they act in a common pathway. Studies from our laboratory have shown that NPC2 is a cholesterol transporter, and that the unique lysosomal phospholipid lyso-bis phosphatidic acid (LBPA), which is localized to the internal membranes of the LE/LY, causes order of magnitude increases in the rate of sterol transfer by NPC2. For cholesterol to exit across the limiting membrane of the LE/LY, it has been proposed that NPC2 transfers its cholesterol directly to the N-terminal domain of NPC1, although such protein-protein interactions have not yet been experimentally demonstrated. Our recent structure-function studies indicate that multiple domains on the surface of NPC2 are necessary for its cholesterol transport activity. This suggested to us that NPC2 may promote membrane-membrane interactions, and recent in vitro studies show that NPC2, indeed, causes vesicle-vesicle interactions. Thus, our working hypothesis is that NPC2 acts at LBPA-rich membrane contact sites, clearing cholesterol by functioning as a bridge between multivesicular (inner) LE/LY membranes, and that it then delivers sterol to NPC1 in the limiting (outer) LE/LY membrane, by an as-yet unresolved mechanism. The present proposal will therefore focus on defining the mechanisms of cholesterol transport by the NPC2 and NPC1 proteins. Studies in Aim 1 will use targeted mutagenesis to establish the structure-function relationships for NPC2 in cholesterol transport, and a kinetics approach to resolve the mechanism of sterol transfer from NPC2 to NPC1. Aim 2 will explore the molecular mechanisms underlying the dramatic effects of LBPA on sterol transport. The proposed studies will use fluorescence and infrared spectroscopy, as well as biochemical and microscopic approaches, to examine sterol transfer kinetics, protein-membrane interactions, protein-protein interactions, and cholesterol clearance in cells from NPC patients. Overall, the present studies will help enable a molecular level understanding of how the NPC proteins functions in normal cholesterol clearance from the LE/LY, and how NPC2 and LBPA function together in endolysosomal cholesterol transport, thus providing new information about the mechanisms of intracellular cholesterol trafficking.